Application of specific ELISAs for BMP15 and GDF9 to cumulus cell extracts from infertile women.

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific paracrine factors which regulate ovarian cumulus cell (CC) functions. This study aimed to investigate if BMP15 and GDF9 bound to CCs can be characterized, quantified, and show an association with IVF outcomes in infertile women. BMP15 and GDF9 ELISAs were validated and applied to discarded CC extracts. Pooled CCs from individual patients were collected from 120 (cohort 1; BMP15 only) and 81 infertility patients (cohort 2; BMP15 and GDF9) undergoing superovulation. BMP15 and GDF9 levels expressed per CC DNA were correlated with maternal age, clinical and embryology data. Total BMP15 and GDF9 were highly correlated with each other (r = 0.9, p < 0.001). The GDF9:BMP15 ratio was unrelated to oocyte number or age. BMP15/CC DNA and GDF9/CC DNA were unaffected by the type of superovulation and were not related to oocyte/embryo outcomes.

A synopsis of global frontiers in fertility preservation.

Since 2007, the Oncofertility Consortium Annual Conference has brought together a diverse network of individuals from a wide range of backgrounds and professional levels to disseminate emerging basic and clinical research findings in fertility preservation. This network also developed enduring educational materials to accelerate the pace and quality of field-wide scientific communication. Between 2007 and 2019, the Oncofertility Consortium Annual Conference was held as an in-person event in Chicago, IL. The conference attracted approximately 250 attendees each year representing 20 countries around the world. In 2020, however, the COVID-19 pandemic disrupted this paradigm and precluded an in-person meeting. Nevertheless, there remained an undeniable demand for the oncofertility community to convene. To maintain the momentum of the field, the Oncofertility Consortium hosted a day-long virtual meeting on March 5, 2021, with the theme of "Oncofertility Around the Globe" to highlight the diversity of clinical care and translational research that is ongoing around the world in this discipline. This virtual meeting was hosted using the vFairs ® conference platform and allowed over 700 people to participate, many of whom were first-time conference attendees. The agenda featured concurrent sessions from presenters in six continents which provided attendees a complete overview of the field and furthered our mission to create a global community of oncofertility practice. This paper provides a synopsis of talks delivered at this event and highlights the new advances and frontiers in the fields of oncofertility and fertility preservation around the globe from clinical practice and patient-centered efforts to translational research.

Capacitation IVM improves cumulus function and oocyte quality in minimally stimulated mice.

PURPOSE: Oocyte in vitro maturation (IVM) is a patient-friendly reproductive technology but lower success rates than IVF have limited its uptake. Capacitation-IVM (CAPA-IVM) is an innovative new IVM system currently undergoing clinical evaluation. This study aimed to determine temporal effects of the pre-IVM phase of CAPA-IVM on cumulus function and oocyte developmental competence in mildly-stimulated mice. METHODS: Immature cumulus oocyte complexes (COCs) derived from mildly stimulated (23 h PMSG) 28-day-old mice underwent pre-IVM for 0-24 h in medium containing c-type natriuretic peptide (CNP), E2, FSH and insulin, prior to IVM (CAPA-IVM). The effect of pre-IVM duration on cumulus cell function and embryo development post-CAPA-IVM/IVF was assessed. RESULTS: Day 6 blastocyst rate increased incrementally with increasing pre-IVM duration: 40.6 ± 2.0%, 45.8 ± 1.2%, 52.2 ± 3.5%, 53.3 ± 5.9%, and 59.9 ± 2.5% for 0, 2, 6, 12, and 24 h pre-IVM, respectively (P < 0.01). DNA content/COC, a measure of cumulus cell proliferation, was significantly higher with 24 h pre-IVM group compared to 0, 2, or 6 h pre-IVM (P < 0.001). Pre-IVM for 24 h significantly increased cumulus expansion and mRNA expression of matrix genes Has2 and Tnfaip6 and Areg relative to no pre-IVM control (P < 0.01). Cumulus-oocyte gap-junctional communication (GJC) was maintained throughout 24 h pre-IVM (P < 0.0001), and GJC loss was slowed during the subsequent IVM phase, whilst meiotic resumption was accelerated (P < 0.05). Pre-IVM increased COC ATP and ADP content (P < 0.05), but not AMP, ATP/ADP, and energy charge. CONCLUSION: The pre-IVM phase of CAPA-IVM improves the quality of IVM oocytes in a temporally dependent manner and significantly influences cumulus cell function including increased cell proliferation, cumulus expansion, and prolonged cumulus-oocyte GJC.

Non-canonical cyclic AMP SMAD1/5/8 signalling in human granulosa cells.

Development of mammalian ovarian follicles is promoted by the combined action of endocrine cues and paracrine factors. Follicle stimulating hormone (FSH), through the action of cAMP drives follicular growth and development. The oocyte secretes powerful growth factors such as bone morphogenetic protein 15 (BMP15) to regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation through the activation of SMAD1/5/8. This study investigated the role of the cAMP signalling pathway on SMAD1/5/8 action in human granulosa cells. Cyclic AMP enhanced BMP15-induction of a SMAD1/5/8-specific BRE reporter. Moreover, in the absence of BMP ligand, cAMP also activated SMAD1/5/8-induced BRE activity. Cyclic AMP increased canonical downstream targets of BMP signalling such as inhibitor of differentiation (ID) mRNA expression. The observed effects were not mediated by secretion of BMPs as cAMP did not promote BMP ligand mRNA expression and a BMP extracellular antagonist, the BMP type II receptor ectodomain, did not affect cAMP-induced ID mRNA expression. Finally, the ERK1/2 pathway was shown to be required for the maintenance of cAMP-induced SMAD1/5/8 activity. Together our results suggest a novel and non-canonical pathway for cAMP signalling in human granulosa cells. Cyclic AMP appears to promote SMAD1/5/8 pathway activity intracellularly and has the ability to activate canonical SMAD1/5/8 downstream targets. Our results add another layer of complexity to the interactions between endocrine signalling and oocyte-secreted BMP ligands during folliculogenesis. Given the importance of both cAMP and SMAD1/5/8 pathways in follicular development, these interactions are likely required for the fine-tuning of oocyte paracrine signalling by endocrine stimuli.

Multigenerational obesity-induced perturbations in oocyte-secreted factor signalling can be ameliorated by exercise and nicotinamide mononucleotide.

STUDY QUESTION: Can maternal and offspring high-fat diet (HFD)-induced changes in mRNA expression levels in mice be ameliorated by interventions in female offspring? SUMMARY ANSWER: Our results indicate that exercise and nicotinamide mononucleotide (NMN) can ameliorate the negative effects of maternal and post-weaning HFD in female offspring. WHAT IS KNOWN ALREADY: Maternal and post-weaning HFD can perturb offspring developmental trajectories. As rates of maternal obesity are rising globally, there is a need for effective treatments in offspring to ameliorate the negative effects from a maternal obesogenic environment. Modulation of the nicotinamide adenine dinucleotide (NAD+) pathway by exercise and the NAD+ precursor NMN has previously been shown to reduce the effects of obesity. STUDY DESIGN SIZE DURATION: This study consisted of a multigenerational study using C57Bl6 mice. Mice were fed a control (chow) or HFD ad libitum throughout mating, pregnancy and lactation (n = 13-25). Female offspring (n = 72) were then also supplied either a chow or HFD post-weaning. At 9 weeks of age offspring from HFD dams were subjected to exercise on a treadmill for 9 weeks or at 16 weeks of age administered NMN (i.p.) for 2.5 weeks. At 18.5 weeks mice were euthanized and ovaries and cumulus-oocyte complexes (COC) were collected to examine the possibility of ameliorating the negative effects of maternal and post-weaning HFD. PARTICIPANTS/MATERIALS SETTING METHODS: Ovary and COC mRNA expression was analysed using RT-qPCR. An initial screen of candidate genes was developed to test which molecular pathways may be involved in generating adverse reproductive system effects. For histological analysis, ovarian tissue was fixed in paraformaldehyde and embedded in paraffin and stained with haematoxylin and eosin. The numbers of primordial, primary, secondary and antral follicles were counted. MAIN RESULTS AND THE ROLE OF CHANCE: In the offspring's COC, maternal obesity increased both growth differentiation factor 9 (Gdf9: 2-fold; P < 0.05, HFD versus chow) and bone morphogenetic protein 15 (Bmp15: 4-fold; P < 0.05, HFD versus chow) mRNA expression levels while exercise and NMN interventions did not regulate Gdf9 and Bmp15 in the same manner. In whole ovary, maternal diet programmed a 25-50% reduction in FSH receptor and sirtuin-3 mRNA expression levels in daughter ovaries (P < 0.05, HFD versus chow). There was a significant interaction between HFD and intervention on the proportion of large preantral and preovulatory follicles (P < 0.05). However, the increase in preovulatory follicles did not translate to increased oocyte yield. NMN administration resulted in reduced body weight in HFD-fed individuals. LIMITATIONS REASONS FOR CAUTION: It is unclear if the changes in oocyte mRNA expression levels reported here will impact oocyte quality and fertility in offspring. Offspring ovulation rate or fecundity could not be studied here and fertility trials are required to determine if the changes in gene expression do reduce fertility. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate that maternal and offspring HFD perturbs key signalling pathways that are known to regulate fertility in mice, highlighting the importance of interventions in helping to prevent the declining rates of fertility in the context of the current obesity epidemic. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants and fellowships from the National Health and Medical Research Council to R.B.G. (APP1023210, APP1062762, APP1117538) and to M.J.M. and D.A.S. (APP1044295). DAS is a consultant to and inventor on patents licenced to Ovascience, Metrobiotech and GlaxoSmithKline. The other authors declare that there is no conflict of interest.

Oocyte maturation and quality: role of cyclic nucleotides.

The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP - inhibiting the hydrolysis of intra-oocyte cAMP - and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect - the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options.

Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions.

STUDY QUESTION: Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? SUMMARY ANSWER: Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. WHAT IS KNOWN ALREADY: Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. STUDY DESIGN, SIZE, DURATION: This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. MAIN RESULTS AND THE ROLE OF CHANCE: Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P < 0.05), regardless of pre-IVM duration. The final blastocyst yields (proportion of blastocysts/immature oocyte) were 26.3% for the control, compared with 39.2, 35.2 and 34.2%, for the 2, 4 and 6 h pre-IVM FSK + IBMX treatments, respectively. In contrast to standard IVM (control), pre-IVM with cAMP modulators maintained open gap junctions between cumulus cells and oocytes for the duration (6 h) of pre-IVM examined, and persisted for a further 8 h in the IVM phase. Cyclic AMP-modulated pre-IVM increased intra-oocyte GSH levels at the completion of both pre-IVM and IVM, in a pre-IVM duration-dependent manner (P < 0.05), which was ablated when GJC was blocked using CBX (P < 0.05). By 4 h of pre-IVM treatment with cAMP modulators, oocyte H2O2 levels were reduced compared the control (P < 0.05), although this beneficial effect was lost when oocytes were co-treated with BSO. Inhibiting glutathione synthesis with BSO during pre-IVM ablated any positive benefits of cAMP-mediated pre-IVM on oocyte developmental competence (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: It is unclear if the improvement in oocyte antioxidant defence and developmental competence reported here is due to direct transfer of total and/or reduced glutathione from cumulus cells to the oocyte via gap junctions, or whether a GSH synthesis signal and/or amino acid substrates are supplied to the oocyte via gap junctions. Embryo transfer experiments are required to determine if the cAMP-mediated improvement in blastocyst rates leads to improved live birth rates. WIDER IMPLICATIONS OF THE FINDINGS: IVM offers significant benefits to infertile and cancer patients and has the potential to significantly alter ART practice, if IVM efficiency in embryo production could be improved closer to that of conventional IVF (using ovarian hyperstimulation). Pre-IVM with cAMP modulators is a simple and reliable means to improve IVM outcomes. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants and fellowships from the National Health and Medical Research Council of Australia (1007551, 627007, 1008137, 1023210) and by scholarships from the Chinese Scholarship Council (CSC) awarded to H.J.L. and the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad awarded to S.S. The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. We acknowledge partial support from the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Chromosome constitution of human embryos generated after in vitro maturation including 3-isobutyl-1-methylxanthine in the oocyte collection medium.

STUDY QUESTION: Do cleavage-stage embryos obtained from oocytes matured in vitro after pre-incubation with a phosphodiesterase inhibitor (IBMX) carry more chromosomal abnormalities than those generated from oocytes matured in vivo? SUMMARY ANSWER: The rate and type of chromosomal abnormalities in normally developing cleavage-stage embryos generated with an in vitro maturation (IVM) system including pre-incubation with IBMX are not different from those observed in supernumerary embryos obtained from oocytes matured in vivo. WHAT IS KNOWN ALREADY: Very limited information is available about the chromosomal constitution of IVM embryos. Previous studies were carried out using FISH on single biopsied blastomeres or arrested whole embryos and only provided fragmentary information on chromosomal abnormalities in IVM embryos. There is no systematic study of chromosomal abnormalities in all blastomeres of human Day 3 embryos with good morphology. STUDY DESIGN, SIZE, DURATION: Between July 2012 and December 2012, 16 young (age <35 years old) egg donors underwent 18 IVM cycles for the generation of research embryos. Eighteen embryos developed to Day 3 and were analysed using array comparative genomic hybridization (aCGH). PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature oocytes were retrieved from 2 to 10 mm follicles after mild ovarian stimulation with gonadotrophins but without hCG ovulation trigger. At collection, oocytes were pre-incubated with 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor and matured in vitro. After IVM culture, mature oocytes were microinjected with sperm from a single donor. Embryos were cultured to Day 3 after ICSI and all blastomeres of 18 good-morphology embryos were collected individually for aCGH. MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte maturation rate in vitro was 50.2% (120/239). The mean fertilization rate was 68.3% (82/120) and 30.5% (25/82) of fertilized oocytes developed into a morphologically good quality embryo on Day 3 after ICSI. Of these, 18 embryos that developed well up to Day 3 were analysed using aCGH. Eighty of the 123 blastomeres analysed showed at least one chromosomal abnormality. Three out of eighteen embryos had completely normal cells. A single embryo carried a meiotic abnormality, 11 embryos were mosaic and three were chaotic. Although the aneuploidy data of this study are too limited to allow statistical analysis, these data are comparable to our own published data on the chromosome constitution of whole day 3 and day 4 embryos after conventional ART. LIMITATIONS, REASONS FOR CAUTION: Array CGH technology determines relative quantification of chromosomal domains but does not allow for the visualization of chromosomal rearrangements, assessment of ploidy or detection of uniparental isodisomy. Conclusions drawn on segmental abnormalities should be treated with caution. Although the limited number of embryos analysed here precludes firm conclusions, they provide valuable data on possible causes of the reduced potential of IVM embryos. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to describe the complete chromosome complement of all single blastomeres of good-morphology day 3 embryos obtained with IVM (including the presence of IBMX in a pre-incubation medium). The results demonstrate that a high proportion of good-morphology embryos are aneuploid and that there is no obvious increase in aneuploidies as a result of IVM which seems to suggest that the reduced efficiency of IVM technology compared with standard IVF may be accounted for by factors other than aneuploidy, such as cytoplasmic defects or reduced endometrial receptivity. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the TBM (Applied Biomedical Research with Societal Finality) programme of the IWT (Agency for Innovation through Science and Technology - Flanders, 110680) and by a Methusalem grant of the Vrije Universiteit Brussel. C.S. is a post-doctoral fellow of the Fund for Scientific Research Flanders (FWO - Vlaanderen). K.J. is a PhD student funded by the FWO. The University of Adelaide owns a patent family associated with IVM technologies that is licensed to Cook Medical. R.B.G. and J.G.T. are inventors. The remaining authors have no conflict of interest to declare.

Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of heat shock protein 90.

STUDY QUESTION: What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions? SUMMARY ANSWER: Heat shock protein 90 (HSP90) was identified and confirmed as being O-GlcNAcylated in mouse COCs under hyperglycaemic conditions (modelled using glucosamine), causing detrimental outcomes for embryo development. WHAT IS KNOWN ALREADY: O-GlcNAcylation of proteins occurs as a result of increased activity of the hexosamine biosynthesis pathway, which provides substrates for cumulus matrix production during COC maturation, and also for O-GlcNAcylation. COCs matured under hyperglycaemic conditions have decreased developmental competence, mediated at least in part through the mechanism of increased O-GlcNAcylation. STUDY DESIGN, SIZE, DURATION: This study was designed to examine the effect of hyperglycaemic conditions (using the hyperglycaemic mimetic, glucosamine) on O-GlcNAc levels in the mouse COC, and furthermore to identify potential candidate proteins which are targets of this modification, and their roles in oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: COCs from 21-day-old superovulated CBA × C57BL6 F1 hybrid female mice were matured in vitro (IVM). Levels of O-GlcNAcylated proteins, HSP90 and O-GlcNAc transferase (OGT, the enzyme responsible for O-GlcNAcylation) in COCs were measured using western blot, and localization observed using immunocytochemistry. For glycosylated HSP90 levels, and to test OGT-HSP90 interaction, immunoprecipitation was performed prior to western blotting. Embryo development was assessed using in vitro fertilization and embryo culture post-maturation. MAIN RESULTS AND THE ROLE OF CHANCE: Addition of the hyperglycaemic mimetic glucosamine to IVM medium for mouse COCs increased detectable O-GlcNAcylated protein levels (by western blot and immunocytochemistry), and this effect was reversed using an OGT inhibitor (P < 0.05). HSP90 was identified as a target of O-GlcNAcylation in the COC, and inhibition of HSP90 during IVM reversed glucosamine-induced decreases in oocyte developmental competence (P < 0.05). We also demonstrated the novel finding of an association between HSP90 and OGT in COCs, suggesting a possible client-chaperone relationship. LIMITATIONS, REASONS FOR CAUTION: In vitro maturation of COCs was used so that treatment time could be limited to the 17 h of maturation prior to ovulation. Additionally, glucosamine, a hyperglycaemic mimetic, was used because it specifically activates the hexosamine pathway which provides the O-GlcNAc moieties. The results in this study should be confirmed using in vivo models of hyperglycaemia and different HSP90 inhibitors. WIDER IMPLICATIONS OF THE FINDINGS: This study leads to a new understanding of how diabetes influences oocyte competence and provides insight into possible therapeutic interventions based on inhibiting HSP90 to improve oocyte quality. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a programme grant from the National Health and Medical Research Council, Australia, ID 453556. J.G.T. is a recipient of funding from and a consultant to Cook Medical Pty Ltd. The other authors have no conflicts of interest to declare.

Pre-maturation with cAMP modulators in conjunction with EGF-like peptides during in vitro maturation enhances mouse oocyte developmental competence.

Recent studies have independently shown that cyclic adenosine 3'5'-monophosphate (cAMP) modulation prior to in vitro maturation (IVM) and epidermal growth factor (EGF)-like peptide supplementation during IVM improve subsequent oocyte developmental outcomes. This study investigated the effects of an IVM system that incorporates these two concepts. Cumulus-oocyte complexes (COCs) were collected from pre-pubertal mice either 46 hr post-equine chorionic gonadotropin (eCG) (IVM) or post-eCG + post-human chorionic gonadotropin (hCG) stimulation (in vivo maturation; IVV). IVM COCs were treated with the cAMP modulators forskolin and IBMX for 1, 2, or 4 hr (pre-IVM phase) prior to IVM. COCs then underwent IVM with the EGF-like peptides amphiregulin or epiregulin, or with the common IVM stimulants follicle-stimulating hormone (FSH) or EGF. A pre-IVM phase increased the size of the subsequent blastocysts' inner-cell-mass compared to standard IVM, regardless of IVM treatment (P < 0.05). Unlike FSH or EGF, amphiregulin or epiregulin significantly increased blastocyst quality (trophectoderm and total cell numbers) and/or yield (P < 0.01) compared to standard IVM, and were the only treatments that produced blastocysts comparable to IVV-derived blastocysts. Forskolin acutely up-regulated EGF-like peptide mRNA expression after a 2-hr pre-IVM phase (P < 0.001), although EGF receptor and ERK1/2 activities were not significantly different than control. IVV-like levels of EGF-like peptide mRNA expression during IVM were maintained only by supplementing with EGF-like peptides and EGF, since expression levels induced by FSH were significantly lower in vitro than during IVV. However, EGF receptor and ERK1/2 phosphorylation levels were not significantly different across treatment groups. In conclusion, a pre-IVM phase in conjunction with IVM in the presence of EGF-like peptides endows high oocyte developmental competence, as evidenced by increased embryo yield and/or quality relative to FSH and EGF.

Effects of differing oocyte-secreted factors during mouse in vitro maturation on subsequent embryo and fetal development.

PURPOSE: We hypothesised that varying native oocyte-secreted factor (OSF) exposure or using different recombinant OSF peptides would have differential effects on post-in vitro maturation (IVM) embryo and fetal development. METHODS: Mouse cumulus oocyte complexes (COCs) were treated with the purified mature domain of GDF9 and/or BMP15 or were co-cultured with denuded oocytes (DOs) from 0 h or 3 h of IVM. DOs were matured for 3 h as either intact COCs+/-FSH before denuding, or as DOs + FSH. COCs were fertilised and blastocyst development was assessed on days 5 and 6, and either differentially stained for ICM numbers or vitrified/warmed embryos were transferred to recipients to assess implantation and fetal rates. RESULTS: No improvement in embryo development was observed with the addition of GDF9 and/or BMP15 to IVM. In contrast, embryos derived from COCs co-cultured with DOs had significantly improved blastocyst rates and ICM numbers compared to controls (P < 0.05). The highest response was obtained when DOs were first added to COCs at 3 h of IVM, after being pre-treated (0-3 h) as COCs + FSH. Compared to control, co-culture with DOs from 3 h did not affect implantation rates but more than doubled fetal yield (21% vs 48%; P < 0.05). GDF9 Western blot analysis was unable to detect any differences in quantity or form of GDF9 (17 and 65 kDa) in extracts of DO at 0 h or 3 h. CONCLUSIONS: This study provides new knowledge on means to improve oocyte quality in vitro which has the potential to significantly aid human infertility treatment and animal embryo production technologies.

Pioneering contributions by Robert Edwards to oocyte in vitro maturation (IVM).

The history of in vitro maturation (IVM) of mammalian oocytes, especially of human oocytes, holds a special place for Robert Edwards. He was the first to comprehensively examine and demonstrate maturation of human oocytes in vitro and in so doing he changed the course of medicine by fertilizing them in vitro. In reviewing his contribution, we have examined the state of the field at the time and discuss his pioneering insights into mammalian oocyte biology. We will also discuss how some of the major concepts and challenges identified by Edwards 50 years ago remain among the major challenges facing IVM today.

Mode of oocyte maturation affects EGF-like peptide function and oocyte competence.

The function and impact of epidermal growth factor (EGF)-like peptide signalling during ovulation and in vivo oocyte maturation (IVV) has been recently characterized, however, little is currently known about the effect of oocyte in vitro maturation (IVM) on this pathway. The aim of this study was to examine expression and functional aspects of three EGF-like peptides (amphiregulin, epiregulin and betacellulin) and their common receptor (EGFR) in cumulus cells during mouse oocyte IVM compared with IVV. Cumulus-oocyte complexes (COCs) were collected from prepubertal mice either 46 h post-eCG (IVM) or 46 h post-eCG plus 0.5-12 h post-hCG (IVV). Time course experiments showed mRNA expression of all three EGF-like peptides and amphiregulin protein in IVM media were significantly lower for the majority of FSH-supplemented IVM compared with IVV. The supplementation of EGF during IVM yielded EGF-like peptide expression levels comparable with IVV and amphiregulin/epiregulin supplemented IVM. However, despite this, EGF activation of the COC EGFR remained significantly lower at 3 and 6 h of IVM than in vivo, and levels were similar to those observed during FSH-supplemented IVM. The addition of exogenous epiregulin during IVM significantly increased blastocyst rates, and epiregulin and amphiregulin improved blastocyst quality, compared with FSH or EGF. In conclusion, findings from this study suggest that the widely used IVM additives, FSH and EGF, are inadequate propagators of the essential EGF-like peptide signalling cascade. In contrast, the use of epiregulin and/or amphiregulin during IVM leads to improved oocyte developmental competence and therefore may be preferable IVM additives than FSH or EGF.

Effect of varying glucose and glucosamine concentration in vitro on mouse oocyte maturation and developmental competence.

The effects of hyper- and hypo-glycaemic conditions during the in vitro maturation of mouse cumulus-oocyte complexes on developmental competence were examined, with an emphasis on the role of the hexosamine biosynthesis pathway. A low (1 mM) glucose concentration achieved optimal oocyte competence (3-fold higher blastocyst development rate compared with high (30 mM) glucose, P<0.05). In addition, glucose supplementation during only the first hour after release from the follicle was necessary and sufficient to support oocyte maturation and embryo development to the blastocyst stage. Glucosamine (a known hyperglycaemic mimetic and specific activator of the hexosamine pathway) was able to substitute for glucose during this first hour, indicating that flux through the hexosamine pathway is essential for oocyte competence. In the absence of glucose throughout the maturation period, glucosamine was not able to increase developmental competence, and at higher concentrations (2.5 and 5 mM) had a detrimental effect on MII and blastocyst development rates, compared with controls (P<0.05). These experiments underscore the importance of glucose metabolic pathways during in vitro maturation and support the concept that excess flux through the hexosamine pathway has detrimental consequences.

Regulation of folliculogenesis and the determination of ovulation rate in ruminants.

The paper presents an update of our 1993 model of ovarian follicular development in ruminants, based on knowledge gained from the past 15 years of research. The model addresses the sequence of events from follicular formation in fetal life, through the successive waves of follicular growth and atresia, culminating with the emergence of ovulatory follicles during reproductive cycles. The original concept of five developmental classes of follicles, defined primarily by their responses to gonadotrophins, is retained: primordial, committed, gonadotrophin-responsive, gonadotrophin-dependent and ovulatory follicles. The updated model has more extensive integration of the morphological, molecular and cellular events during folliculogenesis with systemic events in the whole animal. It also incorporates knowledge on factors that influence oocyte quality and the critical roles of the oocyte in regulating follicular development and ovulation rate. The original hypothetical mechanisms determining ovulation rate are retained but with some refinements; the enhanced viability of gonadotrophin-dependent follicles and increases in the number of gonadotrophin-responsive follicles by increases in the throughput of follicles to this stage of growth. Finally, we reexamine how these two mechanisms, which are thought not to be mutually exclusive, appear to account for most of the known genetic and environmental effects on ovulation rate.

Simulated physiological oocyte maturation (SPOM): a novel in vitro maturation system that substantially improves embryo yield and pregnancy outcomes.

BACKGROUND: Oocyte in vitro maturation (IVM) reduces the need for gonadotrophin-induced ovarian hyperstimulation and its associated health risks but the unacceptably low conception/pregnancy rates have limited its clinical uptake. We report the development of a novel in vitro simulated physiological oocyte maturation (SPOM) system. METHODS AND RESULTS: Bovine or mouse cumulus-oocyte complexes (COCs) were treated with cAMP modulators for the first 1-2 h in vitro (pre-IVM), increasing COC cAMP levels ∼100-fold. To maintain oocyte cAMP levels and prevent precocious oocyte maturation, COCs were treated during IVM with an oocyte-specific phosphodiesterase inhibitor and simultaneously induced to mature with FSH. Using SPOM, the pre-IVM and IVM treatments synergized to increase bovine COC gap-junctional communication and slow meiotic progression (both P < 0.05 versus control), extending the normal IVM interval by 6 h in bovine and 4 h in mouse. FSH was required to complete maturation and this required epidermal growth factor signalling. These effects on COC had profound consequences for oocyte developmental potential. In serum-free conditions, SPOM increased bovine blastocyst yield (69 versus 27%) and improved blastocyst quality (184 versus 132 blastomeres; both P < 0.05 versus standard IVM). In mice, SPOM increased (all P < 0.05) blastocyst rate (86 versus 55%; SPOM versus control), implantation rate (53 versus 28%), fetal yield (26 versus 8%) and fetal weight (0.9 versus 0.5 g) to levels matching those of in vivo matured oocytes (conventional IVF). CONCLUSIONS: SPOM is a new approach to IVM, mimicing some characteristics of oocyte maturation in vivo and substantially improving oocyte developmental outcomes. Adaption of SPOM for clinical application should have significant implications for infertility management and bring important benefits to patients.

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus).

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus-oocyte complexes (COCs) were isolated from large (>1.5mm) and small (0.7-1.5mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 +/- 9.3 versus 6.4 +/- 2.7, mean +/- S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.

Metabolism of the bovine cumulus-oocyte complex and influence on subsequent developmental competence.

The two types of cells that make up the cumulus-oocyte complex (i.e. the oocyte and cumulus cells) have very different metabolic demands, with glucose occupying a central role in metabolic activity. Cumulus cells have a significant requirement for and utilise high levels of glucose, yet appear to have little need for oxidative metabolism. In contrast, oocytes have a requirement for oxidative metabolism, although limited glucose metabolism may also be an important aspect of meiotic and developmental competence. Nevertheless, because of the metabolic and communication link between the cumulus and the oocyte, glucose availability and metabolism within the cumulus can have a significant impact on oocyte meiotic and developmental competence. In particular, the role of the hexosamine biosynthesis pathway within cumulus cells appears critical for the supply of substrate from glucose for extracellular matrix production, yet if overstimulated can significantly decrease developmental competence of the oocyte. Current static systems for in vitro maturation are clearly incompatible with meeting substrate demands, especially glucose. In the future, in vitro maturation will include a more dynamic approach, which will adjust nutrient components to meet the changing functional requirements of cumulus-oocyte complexes during the final process of maturation.